The goal of our study was to determine if depletion of gut microbiome diversity would alter host thermal tolerance in a vertebrate ectotherm. We raised laboratory-reared tadpoles of the green frog in two microbial colonization treatments: colonized and depleted. Colonized tadpoles were raised in natural pond water to provide a rich source of microbiota for colonization of the gut, and depleted tadpoles were raised in autoclaved pond water to reduce this source of microbiota. We then performed a suite of experiments to test for differences in thermal physiology between groups. In our first experiment we tested tadpole acute thermal tolerance (critical thermal minimum and maximum). We verified our results in a second set of tadpoles and then tested tadpole survival under heat stress and thermal sensitivity of locomotion performance. Lastly, we tested for differences in physiological traits at lower levels of biological organization to identify putative mechanisms behind our results including mitochondrial enzyme activities, phospholipid membrane composition, and whole-organism metabolic rate.